Currently, high-throughput sequencing is one of the important research means in molecular biology, medical diagnosis, among other fields. Ever since the birth of the high-throughput second-generation sequencing technologies, rapid developments have been achieved in sequencing technologies. The sequencing cost in the second-generation sequencing has decreased by several orders of magnitude in comparison to the expensive sequencing cost in the past, and the sequencing time has been greatly shortened. Now, the emergence of the third-generation sequencer makes the competition of the sequencing markets even more intense and the development even more rapid. Therefore, every sequencing company or research group has to seek to reduce sequencing cost, shorten procedure time and increase result accuracy in order to take the lead in competition.
Among the second-generation sequencing platforms, a sequencing platform with a high precision and a high sequencing through-put, developed by Complete Genomics Corporation (CG), has a data accuracy of up to 99.9998%, providing accurate gene information for cancer research, detection of low-frequency mutations, and personal genome sequencing. However, the procedure time of library construction associated with the CG platform is too long, the cost of library construction is high, and the library insert fragments are short, limiting subsequent data generation and analysis. This will not only affect the progress of scientific research projects, but also hinder the use and development of the CG platform on a large scale and in wide areas. The library construction process must be optimized, the library construction time shortened, the library length increased and the cost lowered in order for the CG platform to maintain competitive advantage in the intense competitions at present. In the conventional library construction procedure with the CG platform, the steps from double strand cyclization to single strand cyclization at last are rather tedious. The library construction procedure mainly includes 12 steps: enzyme digestion of cyclic DNA, dephosphorylation, end repairing, ligation of 3′ end adaptor, ligation of 5′ end adaptor, gap translation, polymerase chain reaction, single strand separation, cyclization, among other steps, and purification using magnetic beads needs to be performed following each enzymatic reaction. Such a procedure is long and costly. The library insert fragments produced by enzyme digestion are only 26 bp. This is not only unfavorable to the application of the CG platform on a large scale, but also inconsistent with the requirements of CG next-generation sequencing platforms with respect to fragments.